| Objective:Liposarcoma represent the most common adult malignant soft tissue tumor,and could be divided into four major subtypes: atypical lipomatous tumor/well-differentiated liposarcoma(ALT/WDL)and dedifferentiated liposarcoma(DDL);myxoid liposarcoma(ML);pleomorphic liposarcoma(PL);myxoid pleomorphic liposarcoma(MPL).The ALT/WDL/DDL represent as the most common subtype of liposarcoma,and it count for 50-60% of all liposarcoma.ALT/WDL usually seen in the deep soft tissue of extremities and trunk,and followed by retroperitoneum and paratestis.The ALT termed as tumors which located in peripheral parts such as limbs and can be easily removed completely,and the WDL termed as tumors occurred at central site,such as retroperitoneum,mediastinum,spermatic cord,etc.,and difficult for operation.ALT/WDL is prone to relapse,but rarely metastasize.DDL is considered to be a tumor homologous to ALT/WDL and mainly seen in retroperitoneum,it representing a malignant neoplasm with higher recurrence rate and metastasis possibility,with a 5-year survival rate of as low as 40%.About 90% of DDL were de novo,only 10% were developed from ALT/WDL.The main treatment approach for ALT/WDL/DDL is the surgical resection,while it showed insensitivity to traditional radiotherapy and chemotherapy,thus the prognosis of the tumor which located in central site is poor.Therefore,for the treatment prospects of ALT/WDL/DDL,further studies in terms of pathogenesis,exploring and verifying possible therapeutic targets are of great importance.There have been studies(including studies of this group)indicated that ALT/WDL/DDL is characterized by giant ring chromosomes,which consist of12q13-15 amplification,including MDM2,CDK4,CPM and HMGA2 etc.,while the targeted inhibitors for these genes showed not so effective in existing studies.The result of clinical trials of MDM2 gene targeted antagonist has shown not ideal.And the inhibitor of CDK4 have been approved for clinical treatment,while the effect is still limited and needs time to verify.For this reason,keeping explore other molecules or pathways involved in the pathogenesis of ALT/WDL/DDL and discovery of potential therapeutic targets are significant.Our research group has a long-term cooperation with Mayo Clinic,one research finding that FRS2 is another amplified gene located in 12q13-15 region in ALT/WDL/DDL for the first time.In our subsequent independent research,we confirmed the amplification rate of FRS2 in ALT/WDL/DDL was 93.2% by performing fluorescence in situ hybridization(FISH)on large series(146 cases of ALT/WDL/DDL in Chinese population).Which indicated that FRS2 is another consistently amplification gene in ALT/WDL/DDL.FRS2 gene encodes fibroblast growth factor receptor substrates 2(FRS2)protein,which is the “center” adaptor of fibroblast growth factor receptor(FGFR)and can further stimulate the downstream PI3K/AKT and MEK/ERK pathways after being activated,thereby exerting corresponding functions such as affecting tumor proliferation and differentiation.The FGFR family contain four homologous isomers.Among them,FGFR1 is the most famous one in soft tissue tumors and most closely associated with FRS2,so this study focused on FGFR1.There was no inhibitor direct effect on FRS2,while several inhibitors related to FGFR and its downstream pathway have been approved for the treatment of other malignant tumors by food and drug administration(FDA).Besides,in other studies of FGFR activation-related tumors,it was found that inhibiting FGFR can inhibit FRS2 and downstream molecules thus effect on tumor.Thus,inhibiting the upstream and downstream pathway molecules of FRS2 may become a new direction for molecular targeted therapy of this tumor.However,there are few studies focus on FGFR1/FRS2 pathway and its downstream pathways in liposarcoma.In the previous study of our research group,it was found that the detected six DDL cell lines had different levels of high expression of phosphorylated FRS2 protein compared with normal adipocytes,and the positive rate of phosphorylated FRS2 was found to be 90.8% in 197 clinical ALT/WDL/DDL samples,suggesting that FGFR1/FRS2 is activated in this tumor.The literature results also suggest that there is activation of FGFR1/FRS2 pathway in ALT/WDL/DDL.However,the use of FGFR inhibitors alone in vitro is effective but short-lived,and another study has shown that some ALT/WDL/DDL cell lines were insensitive to FGFR inhibitors to FGFR inhibitors.These above results suggest that there may exist other pathways also involved in the pathogenesis of ALT/WDL/DDL besides FGFR1/FRS2 pathway,researchers have also aware of this phenomenon but not studied it further.As one of the upstream pathway molecules of FRS2,EGFR has been well studied in epithelial malignant tumors.It has been found that it can stimulate the phosphorylation of FRS2 and to play corresponding role.However,in soft tissue tumors,especially liposarcoma,there were few studies of the activation of EGFR and its mechanism.Previous studies pointed out that 15% of ALT/WDL/DDL cases had EGFR amplification,and found that EGFR protein high expressed in ALT/WDL/DDL.In addition,one research found activation and high expression of EGFR in another subtype of liposarcoma(myxoid liposarcoma).In the early stage of this study,the test result of ALT/WDL/DDL clinical samples found that in addition to FGFR1,another upstream molecule of FRS2,epidermal growth factor receptor(EGFR),also has high expression of RNA level.And in ALT/WDL/DDL cell lines,the abnormally elevated expression of phosphorylated EGFR after FGFR inhibitor treatment suggests that EGFR can be feedbackally activated in this tumor after the FGFR1/FRS2 pathway is inhibited.Therefore,this study speculates that in ALT/WDL/DDL,the EGFR pathway and the FGFR1/FRS2 pathway can interact each other and play a common role in the tumorigenesis and development of this entity.The role of EGFR pathway and whether it possess interaction with FGFR1/FRS2 pathway in liposarcoma,especially the ALT/WDL/DDL,need to be further investigate.There is no research focus on the interaction between EGFR and FGFR in liposarcoma.While in the studies of epithelial-origin tumor,such as non-small cell lung cancer,breast cancer,bladder cancer,etc.,the result showed that FGFR and EGFR pathways interact and regulate each other,participate in the tumorigenesis and drug resistance,and related to prognosis and treatment together.Besides,these results showed that the combined inhibition of both EGFR and FGFR can effectively prevent the drug resistance caused by the use of a single inhibitor.It is proved that the combined inhibition of the two pathways can exert a better tumor suppressor effect than a single inhibition.Based on the above findings,we speculate that in ALT/WDL/DDL,FGFR1 pathway and EGFR pathway play an important role in tumor growth together.And after FGFR1 pathway is inhibited,EGFR may be activated abnormally,replacing FGFR1 to activate FRS2 and its downstream pathways again to result suppression escape.Co-inhibition of FGFR1 and EGFR pathways in ALT/WDL/DDL is hopeful to overcome the limitation of FGFR1 inhibitor,and to obtain durable and effective tumor suppression.ALT/WDL/DDL has its own unique clinical and genetic characteristics,it can be accurately diagnosed and distinguished through genetic testing.However,most basic researches have not carried out comprehensive genetic verification or even any verification of the included samples,and the cell lines used have not been tested and classified.It can even be seen that the same cell line used as different subtype of liposarcoma in different studies.This study will implement strict criteria for inclusion of experimental materials.The clinical samples were included after histomorphological review and genetic testing.At the same time,multi-platform genetic testing will be performed on the cell lines used in this study for accurate classification and to ensure the accuracy.This study aims to explore the interaction between FGFR1/FRS2 and EGFR pathways in ALT/WDL/DDL and its regulation of downstream molecules,and provide theoretical basis and experimental clues for the early realization of clinical targeted therapy of ALT/WDL/DDL.The research will be conducted from the following aspects: 1)Strict inclusion criteria of experimental materials;2)Systematic detection of the expression of FGFR1/FRS2 and EGFR pathway molecules in ALT/WDL/DDL;3)Detection the inhibition of exogenous inhibitors for ALT/WDL/DDL in vivo and in vitro,and compare whether the combined inhibition of the two pathways can achieve better tumor suppression effects;4)The role of FGFR1 and EGFR in the occurrence and development of ALT/WDL/DDL tumors.Materials and Methods:1 Systematic detection of the activation of the main signal molecules in the FGFR1/FRS2 and EGFR pathways in ALT/WDL/DDL:(1)Screening out the recent samples with good quality,and expand the sample size of detection FRS2 amplification by fluorescence in situ hybridization(FISH)in ALT/WDL/DDL,to further confirm the amplificated rate in the tumor,performing MDM2/CDK4 amplification test at the same time.(2)Detecting the expression level of downstream core molecule AKT and ERK proteins by immunohistochemistry(IHC).(3)Extensive detection of the RNA expression level of FRS2 and its upstream and downstream molecular by real-time quantitative reverse transcription polymerase chain reaction(qRT-PCR),the detected molecular including AKT,ERK,FGFR1,EGFR,FRS2,PI3 KCA,KRAS,MEK,STAT3,IGF1,CDK4,MDM2,BRAF,GRB2,JUN,PTEN.(4)Detecting the DNA expression level of FGFR1 and EGFR by FISH and qPCR.(5)Obtaining of cell lines: a)Experimental group: DDL cell line DDL424 was established by primary culture;purchased ALT/WDL cell line 93T449 from ATCC;b)Control group: purchased PL cell line SW872 from ATCC.(6)Performing multi-platform genetic testing on the three cell lines,and perform western blot(WB)detection on the protein expression of FGFR1/FRS2 and EGFR pathway molecules.2 In vitro experiments with exogenous inhibitors to observe whether there is abnormal activation of EGFR after FGFR1 inhibition,and observe the interaction between FGFR1 and EGFR:(1)Firstly,confirm the IC50 value of FGFR specific inhibitor NVP-BGJ-398 and EGFR specific inhibitor gefitinib in each cell line by MTS method;and detect whether there is synergy between the two drugs and determine the concentration of the two drugs after the two drugs work together in proportion.(2)Detecting the expression changes of FRS2 and its upstream and downstream molecules,including FRS2,FGFR1,EGFR,AKT,ERK and their phosphorylated molecules,at different times after using NVP-BGJ-398 by Western Blot(WB),to confirm the effect of NVP-BGJ-398 alone and observe whether EGFR active abnormally.(3)Stimulating cell lines with growth factor(EGF and bFGF),observe the change of upstream and downstream molecular to investigate whether there is an interaction between FGFR1 and EGFR pathways preliminarily.(4)Detecting the effects of these two inhibitors alone and in combination on cell proliferation,migration and invasion,and cell apoptosis in each cell line by the MTS,Transwell and TUNEL assays;and compare the changes of the expression molecules in the pathways between these two treatment by WB.(5)Treating each cell line with two inhibitors and different growth factors(EGF and bFGF),and explore the role of FGFR1 and EGFR in ALT/WDL/DDL cell lines by detection molecules in the pathways.(6)Tumor transplantation tumor experiments in nude mice:Two inhibitors were administrated alone or in combination to observe the effect of the inhibitors alone and in combination on tumor growth,and explore whether FGFR and EGFR could be the potential therapeutic target for ALT/WDL/DDL;3 Establish cell lines with knockdown of FGFR1 and EGFR alone or in combination to further investigate the role of FGFR1 and EGFR in tumor:(1)Confirm the transfection effect,and compare the changes of molecules in the pathways between before and after transfection by WB;(2)Detecting the effects of gene knockdown on cell proliferation,migration and invasion,and cell apoptosis by MTS,Transwell and TUNEL assays;(3)Observe the effect of FGFR1 and EGFR knockdown alone or in combination on tumor growth in vivo by tumor transplantation tumor experiments in nude mice;Results:1.The expression and activation of FRS2,FGFR1,EGFR and downstream AKT and ERK pathways in ALT/WDL/DDL:(1)FISH test for FRS2 amplification: There were 167 cases of ALT/WDL/DDL detected in our research group.FRS2 FISH was performed in 167 ALT/WDL/DDL cases,among which 146 cases(70 ALT/WDL and76 DDL)were analyzed by our group before and the remained 21(12ALT/WDL and 9 DDL).cases were analyzed by the present study.Among these 167 cases,FRS2 amplification were seen in 157 cases,including 75ALT/WDL and 82 DDL,the overall positive rate was 94.0%;the positive rate of ALT/WDL and DDL were 91.5%(75/82)and 96.5%,respectively.All the 103 control cases in previous detection and the 16 control cases in this present research were negative for FRS2 amplification(including normal tissue and other sarcomas).The MDM2/CDK4 FISH test was performed on21 cases of ALT/WDL/DDL and 6 control cases in this present study,and showed all 21 ALT/WDL/DDL were MDM2 amplified(positive rate 100%),there were 3 cases negative for CDK4 amplification among these 18 tested cases(positive rate 83.3%),and all negative CDK4-non amplified cases were ALT/WDL.In ALT/WDL/DDL,FRS2 has an amplification rate which equal to MDM2,and higher than that of CDK4.(2)Immunohistochemical analysis of AKT and ERK: In 197 cases of ALT/WDL/DDL(including 100 cases of ALT/WDL and 97 cases of DDL)and 48 cases of control group,the result showed that the positive expression rate of AKT in WDL and DDL(2+ and above)were 46.0% and 73.2%;the positive expression rates of ERK in WDL and DDL(2+ and above)were64.0% and 80.4%,respectively;the positive rate of AKT in the control group was 16.7%(8/48),the positive rate of ERK was 27.1%(13/48);the positive rate of AKT and ERK in ALT/WDL/DDL were higher than control cases significantly.(3)qPCR test for the expression of pathway molecules: a)ALT/WDL/DDL vs control group: The result showed that compared with the control group(5 cases of normal fat tissue and 7 other tumors,including 3 cases of myxofibrosarcoma and 4 cases of leiomyosarcoma),all the tested molecules,FRS2,AKT,ERK,FGFR1,EGFR,PI3 KCA,KRAS,MEK,STAT3,IGF1,CDK4,MDM2,BRAF,GRB2,JUN,PTEN,showed different degrees of high expression in 47 cases of ALT/WDL/DDL(including 18 cases of WDL and 29 cases of DDL)and the difference was statistically significant(p<0.05);b)ALT/WDL vs DDL: The expression of GRB2,CDK4,EGFR,IGF1 and MDM2 were higher in DDL than in ALT/WDL with statistically significant difference(p<0.05),the expression of BRAF,ERK,MEK and FGFR1 were higher in ALT/WDL than in DDL with statistically significant(p<0.05).(4)qPCR detection of FGFR1 and EGFR expression at the DNA level: The result showed that compared with the control group,both genes are highly expressed in all tested ALT/WDL/DDL.(5)FISH detection of FGFR1 and EGFR: The test performed on ALT/WDL/DDL(FGFR1 tested on 26 cases,including 15 DDL and 11ALT/WDL;EGFR tested on 17 cases,including 10 DDL and 7 ALT/WDL),and the results showed no amplification;all 8 cases in control group also showed no amplification.(6)Identification of cell lines: Both DDL424 and 93T449 cell line contained MDM2 and FRS2 amplification,control PL cell line SW872 was non-amplified of MDM2 and FRS2,and the sequencing result showed that this cell has no gene amplification and has p53 mutation.(7)Expression of important molecules in FGFR1/FRS2 and EGFR pathways in cell lines: Compared with control cell line ADSC(adipose-derived stem cells),both ALT/WDL/DDL cell lines showed high expression of p-FRS2,p-FGFR1,p-AKT and p-ERK to varying degrees,suggesting that there is relative activation of FGFR1/FRS2 pathway in ALT/WDL/DDL.While p-EGFR expression is relatively high in PL cell line SW872.2.The inhibitory effect on tumor in ALT/WDL/DDL of using FGFR1 and EGFR exogenous inhibitors in combination and alone:(1)Determine the concentration of the inhibitors: The IC50 value of 93T449,DDL424 and SW872 cell lines for NVP-BGJ-398 were 1μM,5μM and 6μM,and for gefitinib were 32μM,30μM and 16μM,respectively;the combination index of two inhibitors were <1 in ALT/WDL/DDL cell lines,while no seen of synergy in PL cell line.(2)The change of pathway molecules using FGFR inhibitor alone: After using NVP-BGJ-398,it was observed that the expression of downstream molecules was decreased ALT/WDL/DDL cell lines,especially p-ERK.The expression of p-ERK began to increase after 8 hours,and at the same time it was found in three cell lines p-EGFR is up-regulated to varying degrees after decrease of p-ERK,which is negatively correlated with the expression of p-ERK.This phenomenon can also be observed in the control group PL cell line SW872,but the rebound occurs later and the degree of p-EGFR activation is relatively low.(3)The preliminary detection of interaction between FGFR and EGFR pathways: In the combined treatment experiment of inhibitors and growth factors,it was found that EGF and bFGF can promote the expression of p-FGFR1,p-EGFR and downstream molecules,indicating that the two pathways do indeed have mutual regulation.(4)Cell function experiments: The result show that the inhibition of the two pathways alone or in combination all can significantly affect the migration and invasion ability of the three cell lines,as well as the clonal formation ability.The difference is statistically significant(p<0.05).(5)TUNEL assay: The result showed that in ALT/WDL/DDL cell lines,combination of two inhibitors can promote the increase of apoptotic signal,while there was no difference between these groups of PL cell line.(6)The inhibitory effect on downstream pathway of inhibitors used alone and in combination: Western blot showed that in ALT/WDL/DDL cell lines,compared with inhibiting FGFR1 alone,the combination of the two inhibitors had a stronger inhibitory effect on downstream p-ERK,and no obvious signs of rebound were found during the observation period;while in PL cell line,the combination of two inhibitors showed no significant difference with inhibiting FGFR1 alone,and the phenomenon of inhibition and then rebound can still be observed.(7)Observe the role of pathways and interaction by treat cells by combination of inhibitors and growth factors in different ways: In ALT/WDL/DDL cell lines,compared to inhibiting FGFR1 alone,which is difficult to completely inhibit the effects of EGF and bFGF,the two inhibitors used in combination can effectively inhibit the stimulation of growth factors on p-FGFR1,p-EGFR,p-AKT and p-ERK.While in PL cell line,the combination of the two inhibitors still not achieved complete inhibition of p-EGFR and p-ERK.(8)The tumor formation experiments in nude mice: The result showed that93T449 cells are difficult to form tumors due to the low degree of malignancy.In DDL424 formed tumors,gefitinib can produce a certain inhibitory effect,and NVP-BGJ-398 can significantly inhibit tumor growth;besides,compared with NVP-BGJ-398 alone,the combination also has a better and obvious tumor inhibitory effect in DDL424 formed tumors.While in PL cell line formed tumor,neither of the two inhibitors used individually or in combination had significant tumor inhibition effects.3.The effect of knockdown of FGFR1 and EGFR in ALT/WDL/DDL:(1)The change of pathway molecules after transfection: The overall change trend of the three cell lines after transfection was similar,the downstream molecules were decreased after knockdown of both FGFR1 and EGFR.The expression of FGFR1 was increased after knockdown of EGFR and vice versa,which indicated that there were mutual regulation and compensation between two pathways.(2)MTS assay: In ALT/WDL and PL cell line,knockdown FGFR1 and EGFR in combination by lentiviral transfection could decrease the cell proliferation.In DDL424,knockdown of EGFR and FGFR1 individually or in combination could all reduce the cell proliferation.(3)Transwell assay: In ALT/WDL/DDL cell lines,knocking down FGFR1 and EGFR alone and in combination can induce the cell ability of migration and invasion.The difference is statistically significant(p<0.05).The degree of inhibition from strong to weak is combination>FGFR1>EGFR.While in PL cell line,knocking down FGFR1 and EGFR alone and in combination can induce the cell ability of migration and invasion.The difference is statistically significant(p<0.05).The degree of inhibition from strong to weak is combination>FGFR1>EGFR.(4)Clone formation experiments: The result showed that in ALT/WDL cell line,knockdown of genes individually or in combination had no significant effect on the ability of cell clone formation.In DDL424,knockdown of FGFR1 and double genes could reduce cell clone formation.In PL cell line,knockdown of EGFR could affect cell clone formation.(5)TUNEL assay:In ALT/WDL cell line,the increase of apoptotic signal could be seen in all groups,while there were on difference between groups in DDL424 and PL cell line.(6)The tumor formation experiments in nude mice: In vivo,EGFR knockdown and FGFR knockdown in DDL424 can affect tumor formation to varying degrees,but double gene knockdown is more obvious with significant difference(p<0.05).While in PL cell line formed tumor,FGFR1 knockdown does not affect tumor formation,while EGFR knockdown can significantly inhibit tumor growth and the tumor were smaller in double gene knockdown with significant difference(p<0.05).Conclusion:1.The amplification rate of FRS2 in ALT/WDL/DDL in our research group is94.0%,its amplification ratio is slightly lower than the most famous driver gene MDM2 in 12q13-15,and higher than another famous amplificated gene CDK4.It reminds that FRS2 represented as another driver gene in this entity.Combined detection of FRS2 amplification will assist clinical differential diagnosis of ALT/WDL/DDL.2.In ALT/WDL/DDL,FGFR1/FRS2 and EGFR pathways are activated.EGFR pathways and FGFR1 pathways can interact and give feedback each other.FGFR1 and EGFR have important roles in tumor growth and participate in tumor occurrence and development.3.Inhibition of FGFR1/FRS2 has a short and limited inhibitory effect on ALT/WDL/DDL cells.EGFR is abnormally activated after FGFR1 inhibition,suggesting that EGFR is activated after FGFR1 inhibition to replace the effect of FGFR1 to activate the expression of downstream molecules.4.Compared with the inhibition of FGFR1 alone,the combined inhibition of FGFR1 and EGFR can achieve a more effective and lasting inhibitory effect on ALT/WDL/DDL tumor cells.5.At present,there are few in vivo studies in ALT/WDL/DDL researches.This study can provide important in vivo experimental data for tumor targeted therapy research.6.The results of the control group study suggest that EGFR may play an important role in the occurrence and development of PL,and it is worthy of further discussion.It may provide new research ideas for this highly aggressive subtype of liposarcoma,which no characteristic genetic changes have been found.7.The accuracy of experimental materials is an important guarantee for obtaining credible results.At some present studies of liposarcoma,sample inclusion was not standardized enough.In this study,the experimental materials have been verified by histopathology and multi-platform genetics,providing a reliable preliminary stage for this study basis.The identification of cell lines,especially the accurate classification of PL cell lines,also provides important clues for future research on this subtype. |
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